Parasites of the trypanosomatid protozoan genus Leishmania are responsible for a spectrum of tropical diseases that afflict more than 10 million people worldwide, and depending on the specific species and immune status of the infected person, can be severe or fatal. In Brasil, Africa and the Mediterranean basin, leishmaniasis is a common opportunistic infection in AIDS patients, and many US soldiers now stationed in the Middle East have been infected. The goal of our research is to develop and apply new methods for the identification of genes used by this parasite to carry out its infectious cycle. Our premise is that the availability of powerful molecular genetic tools, and identification of genes important to parasite virulence, will radically advance our ability to develop improved control strategies. In previous studies powerful new methods were successfully developed for manipulating the parasite genome in a variety of ways, and modern 'genomic approaches' such as expression profiling and comparative genome hybridization applied. These studies were quite successful, but the number of exciting genes identified far exceeded the ability of our methods to tackle them. Fortunately we achieved two breakthroughs. First we showed that the RNA interference pathway was functional in species belong to the L. braziliensis species complex, despite its loss in other Leishmania species. We showed that the RNAi pathway could be used to conveniently and rapidly adapted to functionally assess gene function within the parasite. The features of this methodology promise to allow us to rapidly scale up and test many genes, and determine their role in virulence. The loss of this pathway in some Leishmania species is provocative and our studies suggest this is linked to important changes in the infectious cycle of great relevance to human disease. Our second breakthrough was the demonstration that it was possible to generate crosses of Leishmania experimentally. These new approaches will allow us to better describe the functional organization of the Leishmania genome, and by studying the inheritance of traits affecting animal virulence, to identify genes relevant to human disease in ways not previously possible. PUBLIC HEALTH RELEVANCE: Leishmania are important tropical parasites, causing disease in more than 10 million people worldwide; more than 400 million people are at risk for infection in endemic regions. US military personnel have significant risk of infection in these areas as well, and recently concerns have risen that leishmaniasis may be endemic in certain dog populations in the USA. Depending on the species, Leishmania-induced pathology ranges from self-healing, cutaneous lesions to fatal, visceral diseases. Currently, there are no vaccines available against leishmaniasis, and the only approved chemotherapies are marginally effective, difficult to administer, and have significant associated toxicities. The underlying tenet of our research program is that improved understanding of key pathways required for parasite virulence and viability may provide opportunities for the development of improved therapies. In this project our goal is to develop and apply new methods for the identification of genes used by this parasite to carry out its infectious cycle. In previous studies powerful methods were successfully developed for manipulating the parasite genome in a variety of ways, and modern 'genomic approaches' such as expression profiling and comparative genome hybridization applied in several ways. These studies were quite successful, but the number of exciting genes identified far exceeded the ability of our methods to tackle them. Fortunately we achieved two breakthroughs. First we showed that the RNA interference pathway was functional in species belong to the L. braziliensis species complex, despite its loss in other Leishmania species. We showed that the RNAi pathway could be used to conveniently and rapidly adapted to functionally assess gene function within the parasite. The features of this methodology promise to allow us to rapidly scale up and test many genes, and determine their role in virulence. The loss of this pathway in some Leishmania species is provocative and our studies suggest this is linked to important changes in the infectious cycle of great relevance to human disease. Our second breakthrough was the demonstration that it was possible to generate crosses of Leishmania experimentally. These studies will allow us to better describe the functional organization of the Leishmania genome, and by studying the inheritance of traits affecting animal virulence in these studies, to identify genes relevant to human disease in ways not previously possible.